Method of skin regeneration using a collagen-glycosaminoglycan matrix and cultured epithelial autograft

ABSTRACT

The present invention relates to a method of skin regeneration of a wound or burn in an animal or human. This method comprises the steps of initially covering the wound with a collagen glycosaminoglycan matrix, allowing infiltration of the grafted GC matrix by mesenchymal cells and blood vessels from healthy underlying tissue and applying a cultured epithelial autograft sheet grown from epidermal cells taken from the animal or human at a wound free site on the animal&#39;s or human&#39;s body surface. The resulting graft has excellent take rates and has the appearance, growth, maturation and differentiation of normal skin.

GOVERNMENT SUPPORT

The research described herein was supported in part by grant numberGMO-7560-17 from the National Institutes of Health. The government hascertain rights in this invention.

BACKGROUND ART

A patient who has suffered extensive skin loss or injury is immediatelythreatened by infection and by excessive loss of fluids. To meet both ofthese needs, a large skin wound must be closed promptly by some type ofmembrane. The most direct method of accomplishing this purpose is bytransplanting partial-thickness sections of skin to the wound, therebysealing the wound and preventing fluid loss and infection.

The transplanted section of skin can be removed ("harvested") from ananimal of another species. This type of transplant is referred to as axenograft. However, a xenograft suffers from the disadvantage that thetransplanted skin is rejected and can only serve to cover the wound forthree to five days. Consequently, a xenograft can only serve as astopgap while the patient's skin slowly heals.

The transplanted section of skin can also be harvested from humancadavers. This type of transplant is referred to as an allograft orhomograft. However, cadaveric skin is in short supply, and allograftsare often, like xenografts, rejected. Although immunosuppressive drugscan increase the period of time which an allograft may cover a wound,they also leave the patient vulnerable to infection. Allografts alsosuffer from the disadvantage that they expose the patient to the risk oftransmission of diseases such as hepatitis and AIDS.

The most desirable form of transplant is an autograft, in which skinfrom an undamaged area of the patient or identical twin is harvested andused to cover the wound. The risk of rejection and disease transmissionis thereby eliminated, and the transplanted skin proliferates to form anew layer of dermis and epidermis.

The harvesting operation is a painful, invasive process, which causesscarring. It should therefore be kept to a minimum. In addition, aseverely burned patient may suffer skin loss or damage on nearly all ofhis or her body. This may severely limit the amount of healthy, intactskin that is available for autografting. When this occurs, xenografts orhomografts may be placed across the entire wound surface to controlinfection and dehydration; they are gradually replaced as autograftsbecome available. Autografts may be harvested repeatedly from a donorsite. In such an operation, an area of xenograft or homograft is removedand discarded, and replaced by an autograft. Each donor site must beallowed to heal before another autograft is removed from it; thisrequires a substantial delay, and prolongs the recovery of the patient.Furthermore, the quality of the skin graft diminishes with eachsuccessive harvest.

Consequently, much effort has been spent to create a skin substitute forthe massively burned patient with limited donor sites. Attempts havebeen made to manufacture artificial skin from both biologic andsynthetic materials with variable results. An acceptable skin substituteshould provide both the components and functional results of normalskin. Two important components of the skin are the epidermis and dermis.The epidermis is the outer layer of skin. It consists of cells atvarious stages of differentiation and maturity. Basal cells are locatedat the lowest level (adjacent to the dermis) and are the leastdifferentiated. The dermis is located below the epidermis and comprisesmesenchymal cells and blood vessels. The junction between the dermis andepidermis is referred to as the basement membrane and is responsible forone of the most important functional results of normal skin, namely thetight adhesion of the dermis to the epidermis. This tight adhesion addsstrength and durability to the skin and prevents "shearing" of theepidermis. "Shearing" is the "rubbing off" of the epidermis when lateralforces are applied to the skin, and can result in blistering and skinfragility.

One of the most promising skin substitutes is a synthetic bilayermembrane (hereinafter collectively referred to as "CG bilayer"). Thismembrane comprises a bottom layer (hereinafter referred to as "CGmatrix") which is a highly porous lattice made of collagen andglycosaminoglycan. The outer layer is a membrane semipermeable tomoisture and impermeable to infectious agents such as bacteria. The CGlattice serves as a supporting or scaffolding structure into which bloodvessels and mesenchymal cells migrate from below the wound, a processreferred to as "infiltration". Infiltration is responsible for creatinga new dermis, referred to as the "neodermis", which replaces the CGmatrix as it biodegrades. Epithelial cells from undamaged skinsurrounding the edges of the wound migrate into CG matrix to create anew epidermis, referred as the "neoepidermis". Because burns and otherskin wounds tend to be shallow, mesenchymal cells need not migrate veryfar to create a neodermis. However, burns often cover large areas of apatient's body surface. Consequently, epithelial cells often mustmigrate great distances to adequately close a wound. As a result, thinskin grafts are required to close the wound. Consequently, a need existsfor new procedures which can hasten the coverage of the CG matrix with aneoepidermal layer.

A second promising technology for manufacturing and applying artificialskin is referred to as cultured epithelial autograft (hereinafterreferred to as "CEA"). In this method split thickness skin samples areharvested from a site on the patient's body surface that is wound free.The epithelial cells from this graft are grown in culture to giveepithelial sheets that are applied directly to the wound bed, basal sidedown.

The CEA method suffers from the limitation that it only applies aneoepidermis to the wound bed. There is no dermis or basement membranepresent at the time of application, and, therefore, no basementmembrane. Thus, there is nothing to secure the neoepidermis to theunderlying tissue, resulting in poor take rates for CEA sheets applieddirectly onto wound beds. This is evidenced by shearing and blisteringof the transplanted CEA. Consequently, efforts have been made to usedermal substrates, such as cadaveric skin to improve take rates.However, allograft rejection, the risk of disease transmission andlimited availability of cadaveric skin are serious limitations on theusefulness of this technique.

Despite the promise of CEA as a technique for treating wounds,improvements are needed if this technique is to adequately meet theneeds of patients with wounds covering large portions of their bodies.Take rates need to be improved without incurring the limitations andrisks involved in using cadaveric skin. Furthermore, a patient's woundsmust either be exposed or temporarily covered during the approximatelythree week period during which the CEA sheets are being grown.

SUMMARY OF THE INVENTION

The present invention pertains to a method for regenerating skin at aburn or wound on a human or animal which comprises the steps of applyinga collagen-glycosaminoglycan matrix (CG matrix) having an outer moisturebarrier (e.g. a silicone layer) to a wound so that the semipermeablemoisture barrier is exposed to the air. Blood vessels and mesenchymalcells are allowed to infiltrate the CG matrix from tissue beneath the CGmatrix, after which the moisture barrier on the CG matrix is replacedwith a cultured epithelial autograft (CEA) sheet. This CEA sheet isproduced by harvesting split-thickness skin samples from an area of thehuman or animal's body surface that is wound free and culturing theepithelial cells from the split thickness skin samples until thecultured cells have reached confluence. Over time a neodermis andneoepidermis are formed at the burn or wound site, resulting in tissuehaving the appearance, differentiation and growth of normal skin.

The present invention has many advantages. It allows immediate woundcoverage, provides excellent CEA take rates and produces a resultsimilar to native skin. Preparation of CEA sheets requires only that aminimal amount of skin be harvested from the patient, thereby reducingthe discomfort to the patient. CG bilayers are completely synthetic andbiodegradable over time. There is therefore no risk of diseasetransmission from donor to patient. In addition, collagenglycosaminoglycan can be manufactured in bulk and stored for extendedperiods of time.

DESCRIPTION OF THE FIGURE

The FIGURE is a time course showing the sequence of steps in the presentinvention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention refers to a method of skin regeneration for burnsor wounds on a human or animal. This invention overcomes many of theshortcomings presented by methods presently used to regenerate skin tocover burns or wounds. The bottom layer is initially a bi-layercomprising a highly porous lattice that is covered with an outermembrane that is a moisture barrier. The wound is covered by this bottomlayer, with the lattice applied directly to the wound and with themoisture barrier exposed to the air.

The lattice serves as a temporary substitute for the dermis and can beany structure that has the following characteristic: the composition andstructure of the lattice must be such that is does not provoke asubstantial immune response from the graft recipient. The lattice mustbe sufficiently porous to permit blood vessels and mesenchymal cellsfrom healthy tissue below the wound to migrate into the lattice. Asdiscussed hereinabove, this migration is referred to as "infiltration"and is responsible for the generation of the neodermis. To facilitatethe formation of the neodermis, the lattice is biodegradable. Thisbiodegradation must not proceed so rapidly that the lattice disappearsbefore sufficient healing occurs, i.e. before sufficient neodermisforms. Lattices that degrade too slowly impede cell migration and causethe formation of a fibrotic layer of cells surrounding the lattice. Alattice which biodegrades after about thirty days is preferable.

The moisture barrier is any composition which can serve as an outersurface to the lattice and must be capable of being manually removed atwill from the lattice when the CEA sheets are to be applied to thewound, as described hereinbelow. Compositions suitable for use as amoisture barrier must also have the property of being semipermeable tothe passage through the wound of fluids from inside the body andimpermeable to microorganisms such as bacteria and viruses from outsidethe body. The moisture barrier also imparts several desirable physicalproperties to the bi-layer such as tensile strength and suturability.

A preferred embodiment of the present invention employs a highly porouslattice comprised of collagen and glycosaminoglycan (referred tohereinafter as "GAG"), i.e. a collagen glycosaminoglycan matrix(referred to hereinafter as "CG matrix") with a silicone elastomer asthe outer membrane. A CG matrix with an outer silicone surface isprepared according to methods known to those skilled in the art. SeeU.S. Pat. Nos. 4,060,081 (Yannas et al., 1977), 4,280,954 (Yannas etal., 1981) and 4,505,266 (Yannas and Burke, 1985), the teachings ofwhich are incorporated herein in their entirety. Various forms of GAGwhich may be suitable for use in this material include chondroitin6-sulfate, chondroitin 4-sulfate, heparin, heparin sulfate, keratansulfate, dermatan sulfate, chitin and chitosan.

It is possible to control several parameters of the CG matrix (primarilycrosslinking density, porosity and GAG content) to control the rate ofbiodegradation of the lattice. Specific conditions for forming a CGmatrix suitable for use in the present invention are given in theExemplification. However, the skilled artisan will know of otherconditions for forming CG matrices with variations of theabove-mentioned parameters which are similarly suitable for use in thepresent invention. In addition, certain applications of skinregeneration may require matrices which degrade more slowly or morequickly. The skilled artisan will be able to recognize applicationswhere it is desirable to vary the properties of the CG matrix, and willbe able to vary the parameters accordingly. The present inventionencompasses such variations in the CG matrix.

Although the research that led to this invention involved CG matricesand silicone outer membranes, the skin regeneration method of thisinvention is not limited to CG matrices and silicone outer membranes.Subsequent research may reveal other fibrous proteins, polymericmolecules or sintered ceramics which can be used in the presentinvention. Such lattices and materials are within the scope of thisinvention.

Once the CG matrix has been prepared, the wound is readied forapplication of the covering. Areas of skin that have been destroyed ordamaged are surgically removed to prevent it from interfering with thehealing process. The entire area of dead and damaged skin is excised, sothat intact epithelial cells are present at the perimeter of the wound.The CG matrix, with the silicone side away from the wound, is drapedacross the wound to avoid the entrapment of air pockets between thewound and the matrix. The membrane is sutured or stapled to the woundusing conventional techniques and then covered with a bandage.

After application of the CG bi-layer, blood vessels and mesenchymalcells from underlying healthy tissue begin, as described hereinabove,the process of infiltration of the grafted CG matrix. "Infiltration", asdefined herein, further refers to allowing a sufficient period of timefor this migration of mesenchymal cells and blood vessels. Sufficienttime periods are those which permit subsequent application of CEA sheetswith nearly complete take rates, as described hereinbelow. A preferredperiod of time is about ten days, but periods as low as about two tothree days can also be used.

Another aspect of the present invention refers the regeneration of skinat a burn or wound site on a human or animal using seeded CG matrices."Seeded CG matrices" refer to CG matrices into which epidermal or dermalcells harvested from a wound free site on the patient's body surfacehave been introduced. Each epidermal or dermal cell that survives theseeding process can reproduce and multiply, thereby hastening theformation of a neoepidermis and/or neodermis. Preferred epidermal anddermal cells are keratinocytes and fibroblasts, respectively. Seeded CGmatrices are described in U.S. Pat. No. 4,060,081, the teachings ofwhich are incorporated herein by reference in its entirety. Matriceswhich have been seeded are referred to as "cellular" while unseededmatrices are referred to as "acellular".

Seeded CG matrices may be autologous, i.e. matrices seeded with cellsobtained from the human or animal having the burn or wound, or they maybe heterologous, i.e. seeded with cells obtained from a donor. Inaddition, cells being used to seed a CG matrix may undergo geneticmanipulation in order to prevent rejection or to change the cell'sphenotype in some beneficial manner. Genetic manipulation includesintroducing genetic matter into the cells so that the protein geneproduct or products are expressed in sufficient quantities to cause thedesired change in phenotype. An example of suitable genetic matterincludes the gene encoding for a growth factor along with the requisitecontrol elements.

Once infiltration has occurred the wound is ready for the application ofa CEA sheet onto the CG matrix. The CEA sheet eventually forms aneoepidermis. The CEA sheets are prepared by surgically removingsplit-thickness skin samples from the patient in an area of thepatient's body surface (hereinafter referred to as the "donor site")that is wound free. This procedure can take place prior to, concurrentwith or after the application of the CG bilayer to the wound bed. Theepidermis is enzymatically and/or mechanically removed from the dermis.The epidermal layer is then mechanically and/or enzymatically separatedinto small pieces of epidermis and preferably into individual cells. Theepidermal cells are then placed in culture media and allowed toreproduce until all available intercellular space has been eliminatedand the cells have formed sheets from about one to seven layers thick.Cultured epithelial cells that have reached this stage are said to have"reached confluence". This process takes approximately three weeks. Theepithelial cells may be subcultured a number of times to produceadditional cultured grafts. Subculture takes a primary CEA, separatesand resuspends individual cells to be recultured. This can allow forincreases of up to four orders of magnitude in areas which can becovered by cultured grafts. A specific procedure for preparing CEA isincluded in the Exemplification. Preparations of CEA are well known inthe art, and the skilled practitioner will know of many variations ofthe specific procedure disclosed in the Exemplification. In addition,there may be certain applications of this invention where variations inthe method of preparing CEA will lead to more desirable results. Theskilled artisan will be able to recognize these applications and makethe appropriate changes. All such variations in the preparation of CEAthat can be used in conjunction with CG matrix to form an artificialskin bilayer are within the scope of this invention. See Teppe, RobertoGeorge Casper, "Cultured Kerotinocyte Grafting: Implications for WoundHealing," Profschrift, Denhang, Netherlands, 1993, the teachings ofwhich are hereby incorporated by references in their entirety.

After CEA sheets have reached confluence, grafting onto the CG matrixcan be performed. The moisture barrier (e.g. silicone outer layer) ismanually removed from the CG matrix. The surface layer of vascularizedmatrix may be excised with a dermatome or surgical blade. Hemostasis isachieved with electrocautery, direct pressure or topical hemostaticagents, if desired.

The CEA sheets are removed from the culture flask. Petroleum impregnatedgauze is secured to the surface of the sheets with surgical clips. Thesheets are then placed on the matrix sites with the basal cell layerside down and secured to the matrix site with sutures or surgicalstaples, thereby keeping the CEA sheet firmly adherent to the matrixsite. The wound is covered with dry sterile gauze which is changedperiodically and the CEA is allowed to adhere. An autograft that "takes"is indicated by visually observing epidermis on the wound surface whichpersists for several days post-grafting. The extent to which a grafttakes can be more precisely determined by the amounts of wound surfacearea which is epithelized, i.e., how much of the wound surface area iscovered by neoepidermis. This can be determined by histological meansand is described more fully in the Exemplification. A graft which takesis typically characterized by the pressure of epithelial cells coveringthe neodermis. Eventually there is a formation of a basement membrane atthe junction of the neoepidermis and dermis, including components suchas anchoring fibrils. This results in a tight union between theneoepidermis and neodermis.

The invention will now be further and specifically described by thefollowing examples.

EXEMPLIFICATION

The effectiveness of the present method of skin regeneration was testedon Yorkshire pigs by applying CEA sheets to four different types ofwounds. In one type of wound a CEA sheet was applied onto a CGsubstrate. A CEA sheet was also applied directly to full thicknesswounds freshly excised to subcutaneous fat and to full thickness woundsfreshly excised to fascia. Excising a wound to fascia refers to a woundin which the epidermis, dermis and subcutaneous fat layers have all beenremoved. The ability of the CEA sheets to take to these three types ofwounds was tested on three animals, each of which had all three types ofwounds. In the fourth wound type, CEA sheets were applied directly to awound seven days after the wound was created by excising to subcutaneousfat. This type of wound, referred to as "granulating", was tested on twoseparate animals which were free from the other wound types.

Split thickness skin samples were harvested from three Yorkshire pigsaccording to the technique described below. The skin samples from eachpig were then cultured separately to prepare CEA sheets according to theprocedure described below. Fourteen days after the harvesting of thesplit-thickness skin samples, 4×4 cm full thickness wounds excised tofat using a surgical scalpel were made on the dorsums of the three pigs.A total of sixteen wounds were made on all three pigs. CG bilayers,prepared according to the procedure described below, were applied to thefreshly excised wounds, silicone side up. The membrane was then securedwith sutures.

Twenty-four days after the taking of the split thickness skin samples,the CEA sheets had reached confluence and were ready for application tothe animals' wounds. In preparation for the CEA grafting, the siliconelayer on the CG matrices that had been grafted onto the wounds of thesubject pigs was manually removed. A total of twelve 4×4 cm fullthickness wounds, using standard surgical techniques, were then made onthe dorsums of the subject pigs. Eight of these wounds were excised tosubcutaneous fat, the other four were excised to faschia.

The CEA sheets prepared from skin harvested from the subject pigs wereremoved from culture and secured to petroleum impregnated gauze withsurgical clips. They were cut to a suitable size and then applieddirectly to the three types of wounds, basal side down. Each animal wasgrafted only with CEA sheets derived from cells harvested from thatparticular animal. Dry sterile gauze was placed over the sheet andsecured to the matrix or wound with sutures or stainless steel staples.Dry sterile gauze dressings were applied and changed daily.

Seven days after the grafting of the CEA sheets, the animals wereexamined for the completeness of graft take. This was first donevisually. Areas of CEA that appeared pink, with a translucent surface,were judged to have taken. How completely the grafts had taken was alsodetermined by quantitative histology. Regularly spaced biopsies weretaken from each wound. The biopsies were spaced so as to berepresentative of the wound area. The neoepidermal was examined alongthe entire cross section of each wound biopsy. Hematoxylin and eosinsections were analyzed along the entire cross section for the presenceof epithelial cells on the neodermal surface. The percentage ofepithelial coverage on the dermal layer is the histological take. Thegross and histological takes are given below in the Table.

The experimental protocol for determining the effectiveness of CEA takewhen CEA sheets are applied to granulating wounds was the same asdescribed hereinabove except for the following modification. Four fullthickness wounds 4×4 cm in size were excised to subcutaneous fat on thedorsum of each animal seventeen days after harvesting the splitthickness skin samples and allowed to granulate. Granulating woundsunderwent periodic dressing changes with a petroleum impregnated gauze.Grafting of the CEA sheets took place seven days later.

HARVESTING OF SKIN

A fasted Yorkshire pig (15-20 kg) is suspended in a Panepinto body slingand anesthetized with 1.0-2.5% Halothane delivered in conjunction with a30:50 mixture of nitrous oxide and oxygen via a facial mask. Apulseoxymeter is used to monitor heart rate and blood oxygen levelsduring the procedure.

The dorsal hair of the scapular area is clipped with shears and theremaining stubble is removed with shaving cream and a razor. The area isthen washed for three minutes with germicidal soap and sterile water. Athree minute application of 70% isopropanol completes the surfacepreparation.

The donor area is then sterile draped and sterile mineral oil is usedfor lubrication. A Goulian knife with a 0.010" shim is used to removestrips of donor skin. The harvested skin is then placed in sterile vialsof phosphate buffered saline (PBS) supplemented with anantibiotic/antimycotic solution for travel to the culture facility.

PREPARATION OF CEA

The skin samples are washed twice in fresh PBS, placed in 0.25% dispasesolution (single layer with no folds or overlap) and either placed at37° C. for two hours of 4° C. over night.

After dispasing, the skin samples are again washed in PBS and theepidermis is mechanically separated from the dermis (with a pair ofdissecting forceps) and placed in a 0.5%/0.01% solution of trypsin andEDTA. The dermis is scraped with a scalpel to remove any basal cells andthen discarded. The epidermal sheets are placed in a single layerwithout overlap; the sheets are then incubated with the trypsin solutionfor 30 minutes to separate the cells.

The epidermal samples are finely shredded with forceps and the cellsolution is resuspended for one minute. The trypsin activity isneutralized by adding an equivalent amount of fetal bovine serum (FBS)supplemented medium (all references to medium refer to 20% FBSsupplemented Waymouth's medium, see below). The cell solution isresuspended for four minutes and filtered through a 100 μm sieve into anew dish. The original dish is washed with 5 ml of medium and filteredto reduce the cell loss during dish transfer. The suspension is placedin a sterile centrifuge tube and centrifuged for five minutes at 1200rpm (5° C.); the dish can again be rinsed with 5 ml of medium todislodge extra cells.

After centrifuging, the supernatant is removed, the pellet isre-suspended in 10 ml of medium, and the cell suspension is placed onice. The cells are counted; trypan blue is used to exclude nonviablecells.

The suspension is then diluted to 1×10⁶ cells/ml and 10 ml of thesolution is plated in 75 cm² culture flasks. The flasks are placed in anincubator (37° C.) with 5% CO₂ and 90% humidity. Culture medium ischanged every other day. Toward the end of the culture period(approximately three weeks), the medium may need changing daily.

CULTURE MEDIUM WITH SUPPLEMENTS

500 ml 1x Waymouth's Medium MB 752/1

114 ml Fetal Bovine Serum (Sigma F-2442)

5 ml 100x MEM Nonessential Amino Acids

2 ml L-arginine Stock (11.4 g/100 ml)

1 ml Sodium Pyruvate (11.0 g/100 ml)

1 ml Putrescine--HCl Stock (116.11 mg/100 ml)

2 ml Insulin (2.5 mg/ml in 4 mM HCl)

1 ml Hydrocortisone (5 mg/ml in 95% EtOH)

10 μl Cholera Toxin (5 mM)

10 ml 100x Antibiotic/Antimycotic (GIBCO)

Amphotericin B (25 ug/ml), Penicillin G Sodium

(10000 units/ml), Streptomycin Sulfate

(10000 ug/ml).

PREPARATION OF CG MATRIX

Bovine hide collagen, 0.5% by weight is dispersed in 0.05M acetic acidand coprecipitate is concentrated by centrifugation and excess aceticacid is decanted. The concentrated coprecipitate is poured into flatstainless steel freezing pans to a volume of 0.3 ml per squarecentimeter and placed on the cooled (-30° C.) shelf of a freeze-drier.The frozen aqueous component of the coprecipitate is sublimated undervacuum to produce a highly porous matrix 2-3 mm thick. The constituentmolecules of the matrix are cross-linked using a 24 hour dehydrothermaltreatment at 105° C. and 30 milliTorr. The now sterile material iscoated with a thin (approximately 0.3 mm thick) layer of silicone, whichis cured in 0.05M acetic acid at room temperature for 24 hours. Thematrix is further cross-linked by a 24 hour treatment with a 0.25% (byvolume) glutaraldehyde solution in 0.05M acetic acid. The ECM analog isthen exhaustively dialyzed in sterile, de-ionized water and stored insterile 70% isopropanol until use. Before grafting, the matrix is rinsedin phosphate buffered saline (PBS) to remove the alcohol.

RESULTS

                  TABLE                                                           ______________________________________                                                       Gross       Histologic                                         Treatment      Take (%)    Take (%)                                           ______________________________________                                        Type I CEA on CG   100    (n = 16)                                                                             97 ± 3                                                                            (n = 2)                               Type II                                                                              CEA on fascia                                                                             0      (n = 4)                                                                              0      (n = 1)                               Type III                                                                             CEA on fat  0      (n = 8)                                                                              8      (n = 1)                               Type IV                                                                              CEA on                    77 ± 10                                                                           (n = 8)                                      granulating                                                                   wounds                                                                 ______________________________________                                         n = number of wounds.                                                    

Gross observations showed complete take of CEA on CG substrate.Histological measurements from 2 sites confirmed that the CEA was 97 ±3%adherent to the CG substrate, by 7 days after grafting. By contrast,gross observation indicated no take of CEA on fascia or fat. This wasconfirmed by an observed histologic take on fat and fascia of 8% and 0%,respectively, by 7 days after grafting. Histological observationindicates that CEA take on granulating wounds (77% ±10) was better thanwith CEA on freshly excised wounds, but still inferior to CEA take on CGmatrix. The ability of CEA to take when applied directly to granulatingwounds has little relevance clinically. A patient suffering from burnsor wounds that cover a large portion of the patient's body surface isfaced with an immediate threat of a loss of body fluids and infection.Consequently, such a patient cannot afford to wait seven days for awound to granulate before closing the wound. Therefore, the improvementrepresented by the present method over existing methods in a clinicallyrelevant setting is determined by comparing the take rates of CEA on CGmatrix with the take rates of CEA on freshly excised wounds. Theseresults indicate that the claimed invention represents a clearimprovement over existing methods of regenerating skin at wound sites.

Growth, maturation and differentiation of the CEA on CG werehistologically similar to that of normal epidermis. CEA grafted on CGmatrix, as compared to on full-thickness wounds, appeared less fragileand seems more resistant to shearing. As early as 7 days after graftingCEA onto CG matrix, the resulting tissue was pink, soft and supple.

EQUIVALENTS

Those skilled in the art will know, or be able to ascertain using nomore than routine experimentation, many equivalents to the specificembodiments of the invention described herein. These and all otherequivalents are intended to be encompassed by the following claims.

The invention claimed is:
 1. A method for regenerating skin at a burn orwound site in a human or animal, comprising the steps of:a) applying toa wound or burn a synthetic bilayer having a biodegradable porouslattice that is covered with an outer membrane that is a moisturebarrier to the wound or burn, wherein the porous lattice is applieddirectly to the wound or burn, and wherein the moisture barrier isexposed to the air; b) allowing the porous lattice to be infiltrated byblood vessels and mesenchymal cells from healthy tissue under the burnor wound site; c) removing the moisture barrier and grafting with aconfluent cultured epithelial autograft (CEA) sheet of geneticallyunmodified cells, wherein the CEA has a basal side which is in contactwith the porous lattice, whereby over time a neodermis and neoepidermisare formed at the burn or wound site, resulting in tissue that have theappearance, growth and differentiation similar to normal skin.
 2. Themethod of claim 1, wherein the bilayer membrane comprises acollagen-glycosaminoglycan matrix (CG) and a silicone elastomer outermoisture barrier.
 3. The method of claim 2 wherein the culturedepithelial autograft is prepared by the method comprising the stepsof:a) harvesting split-thickness skin biopsies from an area of thehuman's or animal's body surface that is free of wounds; b) separatingthe epidermis from the dermis in the harvested split-thickness skinbiopsies; c) growing the separated epidermal cells in culture until theyhave reached confluence.
 4. The method of claim 3, wherein theharvesting of the split-thickness skin biopsies is performedconcurrently with the grafting of the CG matrix.
 5. The method of claim3, wherein the harvesting of the split-thickness skin biopsies areperformed prior to the grafting of the CG matrix.
 6. The method of claim3, wherein the harvesting of the split-thickness skin biopsies areperformed after the grafting the CG matrix.
 7. The method of claim 3,wherein the CG matrix is acellular.
 8. The method of claim 3, whereinthe CG matrix contains autologous or heterologous cells that have beenintroduced into CG matrix prior to the grafting onto the burn or wound.